A great number of diverse pathologies of the organism are caused by disorders in the hemostatic system. Thromboembolic complications developing from diseases such as myocardial infarction, stroke, thrombosis of deep veins or pulmonary artery are among the chief causes of death around the world. Little surprise then that intensive efforts have been going on for years to develop medications that could serve as effective and safe clinical drugs. Above all, these are various antithrombotic agents displaying anticoagulant properties.
Thrombin is the principal enzyme of the blood clotting system converting the soluble plasma protein, fibrinogen, into an insoluble fibrin clot, and, at the same time, setting off a majority of positive and negative feedbacks in the system, activating thrombocytes (platelets), factors V, VIII, and XIII, and also protein C. Thrombin also initiates various cellular and vascular reactions, including proliferation of endothelium cells, ejection of plasminogen activators, and so on. Considering that thrombin is involved in a large number of key bioregulatory events, thrombin inhibition at its active center must be very effective and offer much promise in controlling many patho-physiological conditions.
There are three principal approaches to engage the hemostatic system in order to prevent undesirable thrombosis—using direct and indirect inhibitors of serine proteases in the clotting series (in the first place, thrombin and factor Xa); using antithrombocytic preparations (antagonists GPIIb/IIIa, aspirin, thrombin receptor antagonists, and so on), which reduce the aggregating properties of thrombocytes and, therefore, prevent further clotting; and using vitamin K antagonists, which reduce synthesis of clotting factor precursors in the liver.
Three principal antithrombotic preparations are used in clinics today—unfractionated heparin; oral anticoagulant warfarin (vitamin K antagonist), and aspirin as a thrombocytes aggregation inhibitor. Each of these preparations, however, has limitations in use and produces undesirable side effects.
Unfractionated heparin (UFH) is a natural anionic polysaccharide, a mixture of polysaccharide chains of different lengths constructed from repeating disaccharide units consisting of uronic (L-iduronic and/or D-glucuronic) acid residues and residues of D-glucosamine. Its molecular weight is put differently by different sources at 3,000-5,000 to 30,000-40,000 dalton, peaking at between 12,000 and 15,000 dalton.
Unfractionated heparin and its lighter analogues (low molecular weight heparins, or LMWH) are indirect anticoagulants. They do not inhibit thrombin by themselves, but only enhance the effect of natural plasma clotting inhibitor antithrombin III (ATIII). If, therefore, the ATIII content in a patient's plasma is very low for some reasons, heparins display a weak anticoagulant effect.
Used in clinical practice, unfractionated heparin has a number of failings:                1. Unfractionated heparin has a short-lived effect, which vanishes very quickly after withdrawal from the medication, and UFH cannot lower the risk of repeated thrombotic events.        2. Heparin displays its antithrombotic effect indirectly, and to display it at all, it must be used in the presence of antithrombin in the system        3. Heparin is only active against circulating thrombin, and almost does not inhibit thrombin adsorbed on a clot.        4. An identical dose of heparin has an unpredictable response in different patients for many reasons, including the level of ATIII in plasma, individual preparation injection rate, binding and neutralization of heparin under the effect of different plasma proteins and activated thrombocytes (thrombocytic factor 3, heparinase, and so on). This requires the status of the coagulation system to be monitored at frequent intervals.        5. Risk of hemorrhagic complications and possibility of thrombocytopenia.        6. Osteoporosis is likely to develop as a complication after prolonged heparin therapy (over 6 months) and sufficiently high heparin doses (>15000 units).        7. Heparin can only be injected intravenously in hospital conditions.        
Vitamin K antagonists (of warfarin type) are indirect clotting inhibitors as well. The mechanism of the effect these preparations have on the clotting system is due to their capacity to effectively block synthesis of vitamin K-dependent clotting factors in the liver. Post-translational γ-carboxylation of the N terminal end of a future factor is essential for synthesizing a factor molecule. Carboxylation is absolutely required for factor molecules to be bound (through Ca+2 ions) during clotting to the negatively charged phospholipid surface of activated thrombocytes and fulfill their function. Vitamin K is an important carboxylation cofactor. During the reaction, it alternates between its hydroxy-quinone form, which is actually involved in the carboxylation reaction, and its oxidized epoxy form. Under the effect of vitamin K reductase enzyme, the epoxy form is reduced and can again be drawn into the carboxylation reaction. Preparations in the coumarin group block the reduction.
Warfarin, too, has several limitations and drawbacks. First, there is slow response to the therapy. It is first observed 24 hours after administration, and gathers full strength within several days. Besides, the preparation binds strongly to various food components and is significantly overlapped by many medications. There is also considerable genetic variability in the activity of the warfarin metabolizing enzyme. This explains significant individuals variability in the response to warfarin, suggesting that certain dietary restrictions and systematic monitoring are required for warfarin recipients.
As we said above, antithrombocytic preparations (aspirin, GPIIb/IIIa antagonists, and so on) hinder complete activation of thrombocytes and their contribution to enhancing the clotting reaction, putting constraints on continued production of thrombin. They do not, however, affect the performance of thrombin that has already formed.
To sum up, all the standard antithrombotic agents surveyed above each have their own drawbacks. Some of them are not direct thrombin inhibitors, requiring antithrombin III (UFH or LMWH) to be present in plasma to have effect, or take effect only slowly, inhibiting the synthesis of essential clotting factors (warfarin and so on), while others (antithrombotic agents) do not affect the thrombin already formed. This explains why intensive search has continued for years for “ideal” inhibitors of much the same efficiency and free from many drawbacks of the standard preparations.
Very attractive in this sense is the strategy of developing small synthetic thrombin inhibitors acting as anticoagulants. These inhibitors have a quick and direct effect on thrombin present in blood, inspiring hope of being effective in controlling acute thrombotic complications even in spite of ATIII shortage in plasma.
The strategic search for new direct synthetic serine protease inhibitors in the clotting series is focused on meeting the following requirements such inhibitor is expected to satisfy:                High affinity to the target enzyme (that is, high inhibiting efficiency).        High selectivity in respect of the target enzyme in comparison with other related serine proteases.        Chemical and metabolic stability.        No toxicity.        Weak (or not very strong) binding to plasma proteins.        High bioavailability when administered through the mouth.        Relatively long half-life of the preparation, allowing, if administered orally, the therapeutic level in plasma to be maintained at a level where one or two preparation intakes a day could be enough.        Possibility of simple monitoring of the preparation level.        
Many surveys devoted to the development of low molecular weight thrombin inhibitors have been published by the time of this writing (Shafer J. A., Cardiovascular Chemotherapy: Anticoagulants, Curr. Opin. Chem. Biol., 1998, 2:458-465; Steinmetzer T., Hauptmann J., Sturzebecher J., Advances in the Development of Thrombin Inhibitors, Exp. Opin. Invest. Drugs, 2001, 10(5):845-864; Edmunds J J, Rapundalo S T, Siddiqui M A, Thrombin and Factor Xa Inhibition, Ann. Rep. Med. Chem., 1996, 31:51-60; Wiley M. R., Fisher M. J., Small Molecule Direct Thrombin Inhibitors, Expert Opin. Ther. Patents, 1997, 7:1265-1282; Hauptmann J, Sturzebecher J., Synthetic Inhibitors of Thrombin and Factor Xa: from Bench to Bedside, Thromb. Res., 1999, 93(5):203-241; Vacca J P., New Advances in the Discovery of Thrombin and Factor Xa Inhibitors, Curr. Opin. Chem. Biol., 2000, 4(4):394-400).
Developing medications on the basis of new chemical compounds, however, requires, in addition to assessing their possible pharmacological effect, careful testing of the toxicological properties of the preparations and their possible effect on heredity, and identifying other remote consequences of their application.
This task is complicated by the fact that far from each inhibitor reducing thrombin activity in buffering aqueous solutions can serve as a real anticoagulant to control blood clotting in the organism. This could be related, for example, to the inhibition mechanism. In particular, unless the inhibitor is competitive, enzymatic activity would not be suppressed completely even if all 100% active centers of plasma thrombin are bound to such inhibitor. Residual thrombin activity may be low enough, but in certain cases it cannot be fully suppressed in plasma by the natural thrombin inhibitor—ATIII. This happens because of a certain modification of the thrombin molecule conformation when bound to such inhibitor, which prevents ATIII from approaching the active thrombin center. As a result, blood continues to be exposed to residual thrombin activity for a long time, and the integral coagulation response, far from falling, even can ultimately intensify because of the presence of this compound in the organism. If the prospective thrombin inhibitor reacts with other components of the clotting system (factors or clotting inhibitors), the ultimate response of the system is impossible to predict in advance as well. The strong bond between the inhibitor and various plasma proteins may increase considerably the dose that must be administered to the organism to achieve the desired anticoagulation effect.
With consideration for the aforesaid, it is clear why, with so large a number of synthesized compounds capable of inhibiting thrombin now available, only one—Argatroban, a thrombin inhibitor synthesized in Japan—has passed all necessary tests and has effectively been authorized in clinical practice (U.S. Pat. No. 5,214,052, 1993; Schwarz R. P., The Preclinical and Clinical Pharmacology of Novastan (Argatroban), In: “New Anticoagulants for the Cardiovascular Patient,” Pifarre R., editor, Hanley and Belfus, Inc., Philadelphia, Pa., U.S., 1997, pp. 231-249; Okamoto S, Hijikata A, Kikumoto R, Tonomura S, Hara H, Ninomiya K, Maruyama A, Sugano M, Tamao Y., Potent Inhibition of Thrombin by the Newly Synthesized Arginine Derivative No. 805. The Importance of Stereo-Structure of its Hydrophobic Carboxamide Portion, Biochem. Biophys. Res. Commun. 1981, 101(2):440-446).
The search for new anticoagulants among synthetic low molecular weight thrombin inhibitors continues to present a defiant challenge.
These inhibitors can be used as anticoagulants directly to treat acute thrombotic conditions developing in the organism in consequence of various pathologies.
Besides, they can also be used to forestall hypercoaguability. This invention proposes to use a synthetic low molecular weight thrombin inhibitor displaying anticoagulant activity in plasma, to be added to a standard plasma-substituting solution.
Frequent situations developing in clinic require large quantities of lost blood to be rapidly replaced with artificial plasma-substituting solutions (PSS). Blood loss is caused by injuries, surgeries, sepsis, various obstetric pathologies, disaster medicine, resuscitation, and so on.